Keli Yang

Multiple amino acid substitutions are involved in the adaptation of H9N2 avian influenza virus to mice

To explore adaptation of avian influenza virus to mice we previously performed serial lung-to-lung passages of the influenza A/Chicken/Jiangsu/7/2002 (H9N2) strain, resulting in the isolation of a variant influenza strain lethal for mice. We now report that virulence correlates with improved growth characteristics on mammalian cells and extended tissue tropism in vivo. Sequencing of the complete genomes of the wild-type and mouse-adapted viruses revealed 25 amino acid substitutions. Some were found to reiterate known substitutions in human and swine H9N2 influenza isolates. Functions affected include nuclear localization signals and sites of protein and RNA interaction, while others are known determinants of pathogenicity and host specificity such as the viral polymerase PB2 E627K substitution. These observations suggest that enhanced growth characteristics and modified cell tropism may contribute to increased virulence in mice. We conclude that multiple amino acid substitutions are likely to be involved in the adaptation of H9N2 avian influenza virus to mice.

Characterization of a pathogenic H9N2 influenza A virus isolated from central China in 2007

The entire genome of the A/Chicken/Hubei/C1/2007 (H9N2) virus, isolated from central China in 2007, was completely sequenced and phylogenetically analyzed. Phylogenetic analysis demonstrated that A/Chicken/Hubei/C1/2007 (H9N2) virus represents multiple reassortant lineages, with genes coming from the early mainland China strain (Ck/Bejing/1/94), an H9N2 virus with special genotype (Ck/shanghai/F/98) and other lineages from poultry in Asia. Infection studies indicated that A/Chicken/Hubei/C1/2007 (H9N2) virus replicated efficiently in MDCK cells and in BALB/c mice. The H9N2 virus also replicated to high titers in chicken respiratory tracts and caused overt clinical signs in chickens. Our results suggest that attention should be paid to the natural evolution of H9N2 influenza viruses and to the control of H9N2 influenza viruses in animals.

Transmission of avian H9N2 influenza viruses in a murine model.

Avian H9N2 influenza viruses have circulated widely in domestic poultry around the world, resulting in occasional transmission of virus from infected poultry to humans. However, it is unknown whether H9N2 influenza virus has acquired the ability to be transmitted from human to human. Here, we report that mouse-adapted H9N2 influenza viruses can replicate efficiently and are lethal for several strains of mice. To evaluate the transmissibility of mouse-adapted H9N2 influenza viruses, we carried out transmission studies in mice using both contact and respiratory droplet routes. Our results indicate that mouse-adapted H9N2 influenza viruses can replicate efficiently and be transmitted between mice. This suggests that once H9N2 influenza viruses adapt to new host, they should present potential public health risks, therefore, urgent attention should be paid to H9N2 influenza viruses.

A one-step RT-PCR assay to detect and discriminate porcine reproductive and respiratory syndrome viruses in clinical specimens.

Outbreaks of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) have led to large economic losses and, subsequently, have drawn great attention to its diagnosis and prevention. To facilitate rapid discrimination of HP-PRRSV from classical PRRSV (C-PRRSV), we developed a one-step RT-PCR assay. Primer specificities were evaluated with RNA extracted from 8 viral strains and our results revealed that the primers had a high specificity for PRRSV. The assay sensitivity was 25 copies/μL for both HP-PRRSV and C-PRRSV. A total of 929 serum samples were identified, of which 20.45% were HP-PRRSV-positive and 1.51% were C-PRRSV-positive, which was completely consistent with that of immunochromatochemistry and sequencing method. The proposed assay can detect the virus 2 days prior the onset of symptoms and it can be performed in 2h, thereby providing a rapid method to discriminate HP-PRRSV from C-PRRSV for the identification and prevention of PRRSV infections.

Multiple amino acid variations in the nonstructural proteins of swine Japanese encephalitis virus alter its virulence in mice

In a previous study, we performed serial brain-to-brain passages of swine Japanese encephalitis virus in mice and sequenced the complete genomes of the F5 and F20 passaged mouse-adapted variants. In the current study, we analyzed the differences between their genome sequences and found 12 amino acid substitutions in the nonstructural proteins. We also assessed the growth characteristics of these two variants in mammalian cells in vitro and in vivo. Our investigations revealed that the F20 variant had enhanced growth characteristics and modified virulence compared with the F5 variant. We therefore conclude that multiple amino acid substitutions in the nonstructural proteins of swine Japanese encephalitis virus alter its virulence in mice.

Molecular Characterization of a Highly Pathogenetic Porcine Reproductive and Respiratory Syndrome Virus Variant in Hubei, China

Porcine reproductive and respiratory syndrome virus (PRRSV) has been recognized as one of the most important pathogens of pigs throughout the world. In 2006, more than 10 provinces of China have experienced an epizootic outbreak of pig diseases characterized by high fever, reddened skin and high morbidity and mortality. From June 2006 to April 2007, we have investigated some clinical samples in Hubei province by RT-PCR and cloned several major genes, N, GP5 and NSP2 gene, shown in this study. Phylogenetic analysis of these genes revealed that the highly pathogenic PRRSV variant, ZB, was responsible for 2006 emergent outbreak of pig disease in Hubei province similar with those variants isolated from other provinces in China in 2006, and belongs to the NA-type PRRSV. In the PRRSV variants, the N and GP5 shear about 90% identity with prototypic ATCC VR-2332 and some typical NA-type Chinese isolates, except the 2850bp NSP2 gene (only shares 65% identity with ATCC VR-2332). But they all shear more than and 97% identity with other highly pathogenetic Chinese PRRSV strains. Additionally, there are extensive amino acid (aa) mutations in the GP5 protein and 2 deletions in the Nsp2 protein when compared with the previous isolates. Most of the variants found in 2006 epizootic outbreak of pig diseases in China were the farthest variants from the typical NA-type PRRSV in phylogenetic distance, and these diversities may be responsible for the differences in the pathogenicity observed between these variants and original Chinese PRRSV strains.

Development of sandwich Enzyme-Linked Immunosorbent Assay for the detection of porcine epidemic diarrhea virus in fecal samples

Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea and dehydration in new-born piglets with subsequent economic losses to swine industry. In the current study, gene encoding of 381aa-792aa spike protein (S1) with the main epitope relative to virus neutralization of PEDV was amplified by RT-PCR and inserted into vector pET-30A(+). The plasmid was transferred into Escherichia coli BL21 (DE3). Meanwhile, recombinant protein expression was induced by isopropy1-β-galactopyranoside (IPTG). After denaturation and renaturation of inclusion bodies, the S1 protein was obtained by using purified recombinant S1 protein in immunized female BALB/c mice. Monoclonal antibodies (MAb) against S1 protein, named 4C7 by hybridoma technique were gained successfully. The result showed that MAb can specifically respond to S1 protein and PEDV via ELISA, Western bolt and immunofluorescence assay methods. A sandwich ELISA (S-ELISA) was established by using the captured monoclonal antibodies 4C7. The sensitivity and specificity were compared between S-ELISA and RT-PCR, which showed similar sensitivity and specificity. This work indicated that S-ELISA would be a significant tool alongside a specific diagnostic reagent for PEDV in future.

A Multiplex RT-PCR Assay to Detect and Discriminate Porcine Reproductive and Respiratory Syndrome Viruses in Clinical Specimens

Outbreaks of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) have led to large economic losses in China. The attenuated vaccine (HP-PRRSV JXA1-R) was used to control HP-PRRSV. However, in recent years, co-infection with classical PRRSV (C-PRRSV), HP-PRRSV, and/or HP-PRRSV JXA1-R has been increasing in China, resulting in a significant impact on PRRSV diagnostics and management. To facilitate rapid discrimination of HP-PRRSV JXA1-R from HP-PRRSV and C-PRRSV, a multiplex RT-PCR assay for the visual detection of HP-PRRSV JXA1-R, HP-PRRSV, and C-PRRSV was established and evaluated with reference PRRSV strains and clinical samples. Primer specificities were evaluated with RNA/DNA extracted from 10 viral strains, and our results revealed that the primers had a high specificity for PRRSV. The assay sensitivity was 24 copies/μL for PRRSVs. A total of 516 serum samples were identified, of which 12.21% (63/516) were HP-PRRSV-positive, 2.33% (12/516) were HP-PRRSV JXA1-R-positive, and 1.16% (6/516) were C-PRRSV-positive, respectively, which was completely consistent with the sequencing method. The high specificity, sensitivity, and reliability of the multiplex RT-PCR assay described in this study indicate that it is useful for the rapid and differential diagnosis of HP-PRRSV JXA1-R, HP-PRRSV, and C-PRRSV.